Department of Protein Technology, KTH - Royal Institute of Technology, Stockholm, Sweden and The Donnelly Centre, University of Toronto, 160 College Street, Room 816, Toronto, M5S 3E1, Ontario, Canada
Antibodies are tremendously useful for biotechnological applications, diagnostics and therapy. However, their complex architecture has spurred interest in smaller derivatives such as Fab and scFv that can retain the targeting specificity and be more easily produced. We have constructed two highly diverse (>1010) libraries based on an autonomous human variable heavy (VH) domain. This scaffold was generated by comprehensive mutational analysis of residues in the former light chain interface to identify structurally compatible hydrophilic substitutions that promote autonomous behavior. We integrated a CDRH1 design biased towards Asp aimed to alleviate aggregation problems that are commonly associated with human domain antibodies.
The libraries have been used to select binders to all human Eph receptors, many of which play roles in cancer. Our aim is to use these binders to investigate blocking or activation of specific Eph receptor homo- or heterodimers. In contrast to Fab fragments raised against the same antigens, the domain antibodies typically bind the ligand-binding domain and compete with ligand for binding. We have solved the structure of one EphA1-binder and propose a model for ligand blocking. Furthermore, we have analyzed the influence of CDRH1 charge on stability and aggregation in a panel of EphA1 binders and developed a strategy to CDRH3 to enable selection of heat tolerant clones by phage display.